Antisense oligonucleotides (AONs) are by far the most sequence-specific reagents, which are able to recognize more than ten designated residues in RNA sequences. Therefore, the use of AONs only as probes for gene functions but also as therapeutic agents for inhibition of the initiating events of gene expression as immense biomedical significance. The objectives of the proposed continued research are to elucidate the structure and the stabilities of AONs and their hybrids with RNA as a function of synthetic backbone and/or base modifications and to provide a basis for the structural effects of the single stranded AONs and the target RNA molecules on antisense hybridization. Although only insufficient literature information is present, our ongoing program in the antisense area has provided valuable information concerning the factors that affect the structures and the relative stabilities of a set of AON duplexes containing a single backbone modification. In this proposal, the studies will be extended to includes AON targeting a selected RNA site in human cytomegalovirus (HCMV) gene or loop RNAs, which are models for the loop motifs frequently observed in natural RNAs and in numerous biological functions. DNA dimer synthons containing an important group of achiral backbone, sugar and/or base modifications will be synthesized and incorporated into AONs at multiple positions. Studies will be undertaken using high resolution NMR, structure computation, UV spectroscopy and enzymatic digestion assays. The analyses will focus on: (a) structures and characteristics of AON.RNA hybrids digestion assays. The analyses will focus on: (a) structures and characteristics of AON.RNA hybrids containing the HCMV target and multiply modified AONs, (b) structures and characteristics of the AON.RNA hybrids when located in a loop region and the preferred RNA loop size for a given AON sequence, (c) structures and characteristics of single stranded AONs as a function of their backbone and base modifications and the structural correlations with the nucleolytic stability of these AONs, (d) structures of the single stranded target RNAs and the effects of these single stranded structures on AON.RNA hybridization, (e) the relative thermodynamic stabilities of the antisense duplexes/complexes containing systematic chemical structural variations. In the studies of these complex molecules, we will use site specific nucleobase deuteration, which is an efficient and cost effective isotope labeling method recently reported by this laboratory for the studies of large nucleic acid molecules, as a primary means for NMR spectral simplification. The proposed research complements the current intensive efforts in chemical synthesis and biological experimentation of AONs. The results of these studies should contribute to the fundamental understanding of the antisense principles, to the growing database of diverse nucleic acid structures, and to the development of the next generation of effective antisense gene regulation of effective antisense gene regulation agents for improved treatment of cancer, viral infectious diseases and a wide range of genetic disorders.